Yersinia pestis is the causative agent of the plague and an important bioterrorism threat. Pneumonic plague is readily transmitted from person to person. Thus, a terrorist attack with Yersinia pestis has the potential to rapidly spread and overwhelm any available medical services. Yersinia Yop proteins are key virulence factors that are translocated into the cytoplasm of host cells, where they potently inhibit the immune response enabling Yersinia to grow almost unimpeded in vivo. YopJ inhibits critical signaling pathways used by the host to rapidly activate the immune response, including the NF-kappaB and mitogen activated protein kinase (MAPK) signaling pathways. YopJ is thought to be an ubiquitin-Iike protein protease. It has been proposed that YopJ functions by removing SUMO from an unidentified target protein(s). However, the substrate for YopJ protease activity remains mysterious and the molecular mechanisms by which YopJ inhibits signaling are still unclear. We propose two independent approaches to establish the mechanism(s) used by YopJ to block NF-kappaB and MAPK activation. TRAF6 is a critical component of the signaling pathway which activates both the NF-kappaB and MAPK signaling cascades in response to infection and is an obvious candidate target for YopJ. In the first Aim, the possibility that YopJ deubiquitinates, and thus deactivates TRAF6 will be tested in cell culture experiments and by direct in vitro biochemical assays. Humans and insects, including Drosophila melanogaster, use highly conserved signaling pathways to activate their immune response, including NF-kappaB and MAPKs. YopJ is likely to inhibit the fly immune response. Thus, we propose a second Aim that takes advantage of the enormous power of Drosophila genetics. Using transgenic flies and established genetic techniques, we will analyze the role of the putative deSUMOylating activity of YopJ, and identify the relevant components of the YopJ pathway. By identifying the true targets of YopJ, new therapeutic means to treat the plague can be devised, thus reducing the potential impact of a bioterrorist attack.